CRISPR/Cas9 Genome Editing Technology: A Valuable Tool for Understanding Plant Cell Wall Biosynthesis and Function.

Yuan Zhang,Allan M Showalter

doi:10.3389/fpls.2020.589517

Abstract

For the past 5 years, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has appeared in the molecular biology research spotlight. As a game-changing player in genome editing, CRISPR/Cas9 technology has revolutionized animal research, including medical research and human gene therapy as well as plant science research, particularly for crop improvement. One of the most common applications of CRISPR/Cas9 is to generate genetic knock-out mutants. Recently, several multiplex genome editing approaches utilizing CRISPR/Cas9 were developed and applied in various aspects of plant research. Here we summarize these approaches as they relate to plants, particularly with respect to understanding the biosynthesis and function of the plant cell wall. The plant cell wall is a polysaccharide-rich cell structure that is vital to plant cell formation, growth, and development. Humans are heavily dependent on the byproducts of the plant cell wall such as shelter, food, clothes, and fuel. Genes involved in the assembly of the plant cell wall are often highly redundant. To identify these redundant genes, higher-order knock-out mutants need to be generated, which is conventionally done by genetic crossing. Compared with genetic crossing, CRISPR/Cas9 multi-gene targeting can greatly shorten the process of higher-order mutant generation and screening, which is especially useful to characterize cell wall related genes in plant species that require longer growth time. Moreover, CRISPR/Cas9 makes it possible to knock out genes when null T-DNA mutants are not available or are genetically linked. Because of these advantages, CRISPR/Cas9 is becoming an ideal and indispensable tool to perform functional studies in plant cell wall research. In this review, we provide perspectives on how to design CRISPR/Cas9 to achieve efficient gene editing and multi-gene targeting in plants. We also discuss the recent development of the virus-based CRISPR/Cas9 system and the application of CRISPR/Cas9 to knock in genes. Lastly, we summarized current progress on using CRISPR/Cas9 for the characterization of plant cell wall-related genes.

Highlights

In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome editing system has emerged as a versatile tool to perform precise gene targeting and mutations including gene insertions/deletions, gene replacements, and single base pair conversions (Zong et al, 2017; Soyars et al, 2018; Dong et al, 2020)
Each guide RNA (gRNA) is complementary and binds to a specific target DNA sequence that ends with a short DNA sequence, known as the protospacer adjacent motif (PAM), which is often “NGG.” The PAM region is essential for Cas9 binding and is found 3 bp downstream of the cleavage site of the Cas9 endonuclease (Ran et al, 2013)
We focus on the rationale and principles associated with using CRISPR/Cas9 constructs to generate genetic mutants that disrupt gene/proteins, which are associated with plant cell wall biosynthesis

Summary

Introduction

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome editing system has emerged as a versatile tool to perform precise gene targeting and mutations including gene insertions/deletions, gene replacements, and single base pair conversions (Zong et al, 2017; Soyars et al, 2018; Dong et al, 2020). We focus on the rationale and principles associated with using CRISPR/Cas9 constructs to generate genetic mutants that disrupt gene/proteins, which are associated with plant cell wall biosynthesis. This exception involves using two gRNAs targeting different loci of a gene, where a larger fragment deletion can occur in a CRISPR mutant that can be detected by PCR (i.e., PCR deletion screening) (Figure 3).

Results

Conclusion